1) Field of the Invention
The present invention relates to the production of biofuels, such as butanol, from renewable biomass through a fermentation process using genetically engineered thermophilic microorganisms. Instead of transferring enzyme coding genes in cassettes by recombinant-DNA technology, the present invention involves an entirely new created microorganism resulting from the fusion of two species of Clostridia, to produce a new strains designated C. thermolignocelluloticum. This new life form can carry out fermentation of biomass in a single vessel at relatively thermophilic temperatures (i.e. 45° C. to 65° C.). Use of this microorganism eliminates the need for multistep, multi-vessel, low temperature reaction system and brings about a single vessel system for the direct conversion of lignocellulosic biomass to butanol and other economical important chemicals.
2) Description of Related Art
World oil production capacity reached a peak in 2006 but the demand for oil is continuing to rise. Therefore, it is essential to look for alternative renewable sources of fuel to meet exiting energy demands. In 2007, there were 1.8 million alternative fuel vehicles sold just in the United States, indicating an increasing popularity of alternative fuels. In addition to searching for viable alternate fuels, efforts have been ongoing to reduce the consumption of fossil fuel vehicles. In 2007, there were more than 6.65 million flexible-fuel vehicles (FFVs) on U.S. roads. By the end of 2010, that number increased to nearly 8.1 million. Three U.S. automakers (GM, Chrysler and Ford) have committed to increasing production of FFVs annually. GM, for example, has said that half of all its vehicles could be FFVs by 2012, pending availability of E85 bio fuel. FFVs are capable of operating on E85, which is a blend of 85 percent ethanol and 15 percent gasoline. Ethanol is a common ingredient in most gasoline formulations as well, but is only used in quantities of 5 to 10 percent to reduce smog-forming emissions and greenhouse gases.
The increase in E85-capable vehicles has gone hand-in-hand with an increase in the availability of E85 fuel. In March 2008, there were about 2,800 E85 fuel stations in the United States, with an ever-increasing number of additional stations scheduled to come online in the coming years. In contrast, in 2000 there were only 14 E85 stations in the US. However, this is still a small number compared to over 200,000 gas stations in the United States. Accordingly, what is needed is to look for an alternative fuel that uses the same existing 200,000 gas stations and can be used by the current gasoline vehicles without significant alteration. Currently, there are two fuels that fit this definition. One is compressed natural gas (CNG) and another one is Bio-Butanol. The CNG cars are very common in many Asian cities and some parts of Europe where natural gas is abundant. Several automakers will be introducing a duel fuel car that uses oil and CNG simultaneously. Although there are numerous manufacturers that offer factory-built natural gas trucks, step-vans, transit buses and school buses, there are fewer options for consumers who need light-duty cars, vans and pickup trucks. Currently, the only natural gas light-duty vehicle manufactured in the U.S. is the Honda Civic GX. Of note, public transportation across the country has been using CNG for decades. And currently, about 12-15% of public transit buses in the U.S. run on natural gas (either CNG or LNG—liquefied natural gas). That number is growing, with nearly one in five buses on order today slated to run on natural gas. States with the highest consumption of natural gas for transportation are California, New York, Texas, Georgia, Massachusetts and Washington D.C.
With the world's oil supply declining and increasing environmental concerns, there has been a push to develop alternative-fuel vehicles. In addition, to FFV vehicles, hybrid vehicles have become increasingly popular, with more than 200,000 units sold annually in the U.S. in 2005 and 2006 and 350,000 in 2007. Sales will most likely continue to increase as the range of hybrid choices grows: Some 44 hybrids are expected to be on sale by 2012, up from 15 in 2008, according to J. D. Power and Associates Automotive Forecasting. Hybrid vehicle sales are expected to grow from approximately 212,000 vehicles in 2005 to about one million by 2012.
Presently, petroleum is the major source of energy due to its high energy density, easy transportability and relative abundance. About 16% of petroleum, which is not used for energy, is also the essential base material for many chemical products, including plastics, pharmaceuticals, solvents, fertilizers, pesticides, and tar for road constructions. Known oil reserves are typically estimated at around 190 km3 (4.76 trillion barrels). Consumption is currently around 84 million barrels (13.4×106 m3) per day, or 4.9 km3 per year. Therefore, if current demands remain the same, the remaining oil supply will last for about 120 years. Due to increasing demands from developing nations like China, India and Brazil and no increased production, the oil prices are predicted to significantly go up in the coming decades.
Chemically, petroleum is a mixture of a very large number of different hydrocarbons; the most commonly found molecules are linear or branched alkanes, cycloalkanes, aromatic hydrocarbons, or more complicated chemicals like asphaltenes. Each petroleum variety has a unique mix of molecules, which define its physical and chemical properties, like color and viscosity.
The alkanes are saturated hydrocarbons with straight or branched chains which contain only carbon and hydrogen and have the general formula CnH2n+2. They generally have from 5 to 40 carbon atoms per molecule, although trace amounts of shorter or longer molecules may be present in the mixture.
The alkanes from pentane (C5H12) to octane (C8H18) are refined into petrol, the ones from nonane (C9H20) to hexadecane (C16H34) into diesel fuel, kerosene and jet fuel. Alkanes with more than 16 carbon atoms can be refined into fuel oil and lubricating oil. At the heavier end of the range, paraffin wax is an alkane with approximately 25 carbon atoms, while asphalt has 35 and up, although these are usually broken down by modern refineries into more valuable products. The shortest molecules, those with four or fewer carbon atoms, are in a gaseous state at room temperature. They are the petroleum gases.
These different molecules are separated by fractional distillation at an oil refinery to produce petrol, jet fuel, kerosene, and other hydrocarbons. For example, 2,2,4-Trimethylpentane (isooctane), widely used in petrol, has a chemical formula of C8H18 and it reacts with oxygen exothermically. (2C8H18(/)+25O2(g)→16CO2(g)+18H2O(g)+10.86 MJ/mol (of octane)).
The amount of solar energy received at the earth's surface is 2.5×1021 Btu/year, which far exceeds the present human usage of 2.0×1017 Btu/year. The amount of energy from the sun which is stored as carbon via photosynthesis is 10 times the world usage. A significant amount of that form of solar energy captured by photosynthesis and stored in biomass can contribute substantially in conversion of transportation fuel at costs competitive with fossil fuel.
Approximately 70% of plant biomass is locked up in 5- and 6-carbon sugars. These sugars are found in lignocellulosic biomass, which is comprised of mainly cellulose, which is a homologous polymer comprised of long chains of glucose; less so, hemicelluloses which is a heterologous polymer of 5- and 6-carbon sugars; and least of all lignin, which is a complex aromatic polymer. A potentially vast source of renewable energy lies within these lignocellulosic biomass. Each year, more than 40 million tons of inedible plant material, including leaves, stems, and stalks from sources such as corn fiber, corn stover, sugarcane bagasse, rice hulls, woody crops, and forest residues. Also, there are multiple sources of lignocellulosic waste from industrial and agricultural processes, e.g., citrus peel waste, sawdust, paper pulp, industrial waste, municipal solid waste, and paper mill sludge. In addition, dedicated energy crops for biofuels could include perennial grasses such as switchgrass and other forage feedstock such as miscanthus, bermuda grass, elephant grass, etc—much of which is unexploited, and inexpensive and mainly thrown away. Turning this waste lignocellulosic biomass into biofuels, such that it will not interfere with food supplies like the manufacturing of ethanol from corn, is a logical solution.
The woody material that gives plants their rigidity and structure comprises three main types of carbon-based polymer—cellulose, hemicellulose and lignin—collectively called lignocellulosic biomass. When digested via fermentation, these polymers yield chemical components that can be used to make biofuels. Cellulose is a difficult substrate for enzymatic degradation because of its physical properties. Cellulose molecules are composed of chains of β-1,4-linked glucose units. The chains are insoluble and form fibrils in which cellulose chains are arranged in parallel bundles that are very stable due to inter-chain hydrogen bonds and Van der Waals interactions between the pyranose rings. Cellulose is largely insoluble and exists in crystalline microfibrils that make the sugars hard to digest. These cellulose microfibrils are a variety of sugars, making it more complicated to convert to a single product such as ethanol or butanol. The covalent chemical bonds that hold lignin's polymers together make it very difficult to break down. Added to that, the composition of lignin varies from plant to plant, and the true structure of this sturdy material remains unknown.
The microbial degradation of cellulose is carried out by the concerted action of different glycoside hydrolases. According to their mode of action, celluloses are subdivided into endo- and exoglucanases. Endoglucanases randomly cleave the cellulose chains at exposed positions and create new ends, while exoglucanases degrade the polymeric chain from either the reducing or the non-reducing end, producing cellobiose as the main product.
Previously, the best way to break apart these lignocellulosic materials and extract their chemicals for fuel production involves heat and strong chemicals. This is a complex process wherein: once source material has been mechanically ground up, the biomass requires pretreatment using heat, acid or ammonia to rip apart the lignin and expose the cellulose and hemi-cellulose inside. Enzymes can then penetrate the biomass and liberate the sugars, which are then fermented and distilled to produce alcohols.
Methane gas, methanol, ethanol, propanol and butanol are the main types of biofuels that can be considered either to blend with or replace gasoline. Mainly due to already existing technology, ethanol is presently considered the most popular biofuel and is blended with gasoline at concentrations ranging from 1 to 85%. Such mixtures are often referred to as gasohol. However, ethanol use in the US is currently limited due to its unavailability from inadequate production to limited distribution. It is currently available in only a small percentage of US fuel pumps.
Although ethanol is the predominant biofuel at this time, (used as E85 as mentioned above) its use has a number of drawbacks when compared to butanol. The potential quantity of butanol that could be produced from cellulose is over an order of magnitude larger than that producible from corn. In contrast to the corn-to-ethanol conversion, the cellulose-to-butanol route involves little or no contribution to the greenhouse effect and has a clearly positive net energy balance (˜ten times better). Butanol is a four-carbon alcohol and thereby yields 25% more energy than ethanol as measured in Joules/gallon (or liter). This means that its energy output is closer to gasoline and can be used as a replacement for gasoline. Bio-butanol, like ethanol, is produced either from conventional crops, such as corn, or from lignocellulosic feedstock. Some advantages that butanol has over ethanol as a transportation fuel are a higher energy density, which provides more miles traveled per gallon of fuel, and a lower tendency to absorb water, which provides more flexibility for transporting butanol and blending it with gasoline. Unlike ethanol, butanol does not need to be mixed with gasoline for use in internal combustion engines and therefore it can be an adequate substitute for gasoline. Furthermore, butanol production also yields useful byproducts such as hydrogen gas that can be used in fuel cell technology and carbon dioxide and hydrogen than can be marketed as gas for other industrial application such as the production of dry ice and chemicals. Furthermore, butyric acid can be used as a base for producing jet fuel.
Unlike ethanol, butanol can use the existing delivery infrastructure such as tanker trucks, pipelines and service stations. Butanol is less explosive, less evaporative (bp 117° C. as compared to ethanol by of 78° C.) and less corrosive than ethanol and, therefore, safer to handle and distribute. Local/Regional production means less dependence on long-distance fuel supply therefore the systems would be less prone to bioterrorism and more favorable for homeland security.
Butanol was tested in a 1992 Buick without modifying the car engine as 100% gasoline replacement in a 10,000-mile trip across the United States. The fuel mileage of 20-26 miles per gallon of butanol was much better than that for gasoline for the same vehicle that gave 22 miles/gallon. That was over 9% increase. E-Test facilities in 10 states revealed that use of butanol yielded an average reduction of hydrocarbons of 95%, the reduction of carbon monoxide was 97%, oxides of nitrogen were reduced by 37% and the background of carbon dioxide was only 14.7% and carbon monoxide emission was to 0.01%.
To meet current gasoline needs with butanol, it will be necessary to utilize all available biomass including wheat stems, corn stover (the stalks and leaves) and wood shavings from logging, bagasse (sugar cane residues), and other agricultural and forestry biomass described above to produce biofuel. A current disadvantage of butanol versus ethanol is that it is more expensive to produce using existing technology, making it less competitive with ethanol.
Accordingly, it is an object of the present invention to develop a more efficient and cost-effective butanol production process to reduce dependence on fossil fuels.